Biocidal proteins

ABSTRACT

Biocidal proteins isolated from Mirabilis have been characterized. The proteins show a wide range of antifungal activity and are active against gram-positive bacteria. DNA encoding the proteins has been isolated and incorporated into vectors. Plants transformed with this DNA have been produced. The proteins find commercial application as antifungal or antibacterial agents; transformed plants will show increased disease-resistance.

This is a is continuation of application Ser. No. 08/471,329, filed Jun.2, 1995, now U.S. Pat. No. 5,689,048, which is a division of U.S.application Ser. No. 08/117,080, filed Dec. 20, 1993, now U.S. Pat. No.5,482,928.

BACKGROUND OF THE INVENTION

This invention relates to biocidal proteins, processes for theirmanufacture and use, and DNA sequences coding for them. In particular,it relates to antimicrobial proteins isolated from Mirabilis.

Mirabilis comprises about 60 tropical American species, many of whichare cultivated for their ornamental value as garden plants. Mirabilisjalapa is commonly known as "four o'clock" or "marvel of Peru", and haswhite, yellow or red flowers. The tuberous roots of M jalapa are thesource of a purgative drug used as a substitute for jalap.

Although plants normally grow on substrates that are extremely rich infungal organisms, infection remains a rare event. To keep out potentialinvaders, plants produce a wide array of antifungal compounds, either ina constitutive or an inducible manner. The best studied of these arephytoalexins, secondary metabolites with a broad antimicrobial activityspectrum that are specifically synthesised upon perception ofappropriate defence-related signal molecules. The production ofphytoalexins depends on the transcriptional activation of a series ofgenes encoding enzymes of the phytoalexin biosynthetic pathway. Duringthe last decade, however, it has become increasingly clear that someplant proteins can play a more direct role in the control ofphytopathogenic fungi. Several classes of proteins with antifungalproperties have now been identified, including chitinases,beta-1,3-glucanases, ribosome-inactivating proteins, thionins,chitin-binding lectins and zeamatins.

Researchers at Japan Tobacco Inc have previously extracted an anti-viralprotein from Mirabilis jalapa suspension cells (callus initially inducedfrom leaves), and also from root and leaf tissue (Tsutomu Ikeda et al;1987; Plant Cell Reports, 6, 216-218). This "Mirabilis anti-plant viralprotein" (MAP) has a molecular weight of 24 kDa. The amino acid sequenceof MAP has been determined and consists of 250 amino acids. A syntheticMAP gene of 759 base pairs has been cloned into a vector and expressedin Escherichia coli (Noriyuki Habuka et al; 1989; Journal of BiologicalChemistry, 264 (12), 6629-6637). The following patents have beengranted: J88061317 and U.S. Pat. No. 4,701,522 cover the Mirabilis MAPprotein extract; J87027797 covers preparations of MAP by culturingcallus. The following patent applications have also been filed:J63123386 on MAP obtained by cloning callus cells; J02186988 onpreparations of the anti-viral protein by culturing E colitransformants; J01294694 on an anti-viral peptide (NOG-22) fromMirabilis, J01294693 on a similar synthetic peptide (NOG-53), andEP414134 on the gene encoding the anti-viral protein. In addition, JapanTobacco have filed patent applications covering two anti-viral proteinsextracted from Bougainvillea (a closely-related genus in the same familyas Mirabilis): BAP-1 has a molecular weight of 33 kDa (J01272598) andBAP-2 has a molecular weight of approximately 30 kDa (J01272599).

We have now purified a new class of potent antimicrobial proteins.

SUMMARY OF THE INVENTION

According to the present invention, we provide antimicrobial proteinscapable of being isolated from seeds of Mirabilis.

In further aspects, this invention comprises a vector containing a DNAsequence coding for a protein according to the invention. The DNA may becloned or transformed into a biological system allowing expression ofthe encoded protein.

The invention also comprises plants transformed with recombinant DNAencoding an antimicrobial protein according to the invention.

The invention also comprises a process of combatting fungi or bacteria,whereby they are exposed to the proteins according to the invention.

A new class of potent antimicrobial proteins has been isolated fromseeds of Mirabilis jalapa. The class includes two protein factors,hereafter called mj-AMP1 (Mirabilis jalapa--Antimicrobial Protein 1) andMj-AMP2 (Mirabilis jalapa--Antimicrobial Protein 2) respectively. Bothare dimeric proteins; Mj-AMP1 consists of two 4 kDa subunits, andMj-AMP2 consists of two 3.5 kDa subunits. Despite their origin, theirprimary structure differs from all other known plant proteins andinstead shows homology to insect neurotoxins found in the venom ofinvertebrates. The amino acid sequence of Mj-AMP1 and of Mj-AMP2 hasbeen determined. These sequences enable manufacture of the protein usinga standard peptide synthesiser.

cDNA encoding Mj-AMP1 and Mj-AMP2 has been isolated and sequenced. ThisDNA can be manufactured using a standard nucleic acid synthesiser, orsuitable probes (derived from the known sequence) can be used to isolatethe actual Mj-AMP gene(s) and control sequences from the plant genome.This genetic material can then be cloned into a biological system whichallows expression of the proteins under the control of a constitutive orinducible promoter. Hence the proteins can be produced in a suitablemicro-organism or cultured cell, extracted and isolated for use.Suitable micro-organisms include Escherichia coli and Pseudomonas.Suitable cells include cultured insect cells and cultured mammaliancells. The DNA can also be transformed by known methods into any plantspecies, so that the antimicrobial proteins are expressed within theplant.

Plant cells according to the invention may be transformed withconstructs of the invention according to a variety of known methods(Agrobacterium Ti plasmids, electroporation, microinjection,microprojectile gun, etc). The transformed cells may then in suitablecases be regenerated into whole plants in which the new nuclear materialis stably incorporated into the genome. Both transformed monocot anddicot plants may be obtained in this way, although the latter areusually more easy to regenerate.

Examples of genetically modified plants according to the presentinvention include: fruit such as tomatoes, mangoes, peaches, apples,pears, strawberries, bananas and melons; field crops such as canola,sunflower, tobacco, sugarbeet, small-grain cereals such as wheat, barleyand rice, maize and cotton: and vegetables such as carrot, lettuce,cabbage and onion.

The Mj-AMP proteins show a wide range of antifungal activity, and arealso active against gram-positive bacteria. The proteins could be usedas fungicides or antibiotics by application to plant parts. Theirantifungal activity is salt-dependent, and may vary with the nature andconcentration of the ions present in the composition. In particular,antifungal activity seems to be decreased by presence of cations. Theproteins could also be used to combat fungal or bacterial disease byexpression within plant bodies.

The Mj-AMP proteins can be isolated and purified from Mirabilis jalapaseeds, synthesised artificially from their known amino acid sequence, orproduced within a suitable micro-organism by expression of recombinantDNA. The proteins may also be expressed within a transgenic plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the gel filtration chromatogram for the antimicrobialproteins and the associated graph of fungal growth inhibition.

FIG. 1B shows the cation exchange chromatogram for the antimicrobialproteins and the associated graph of fungal growth inhibition.

FIG. 2A shows the HPLC profile of purified mj-AMP1.

FIG. 2B shows the HPLC profile of purified Mj-AMP2.

FIG. 3A shows the complete amino acid sequences of Mj-AMP1 (SEQ ID NO:1)and Mj-AMP2 (SEQ ID NO:2); differences are indicated by arrows.

FIG. 3B shows the amino acid sequences of Mj-AMP1 and mj-AMP2, alignedwith those of the neurotoxins.

FIGS. 4A-4H show the time-dependent growth inhibition curves of fungimeasured at varying concentrations of antimicrobial proteins.

FIG. 5 shows the nucleotide sequence (SEQ ID NO:11) and deduced aminoacid sequence (SEQ ID NO:10) of clone MJ1 (Mj-AMP1).

FIG. 6 shows the nucleotide sequence (SEQ ID NO:13) and deduced aminoacid sequence (SEQ ID NO:12) of clone MJ2 (Mj-AMP2).

FIG. 7 shows the construction of the expression vector pMDB1.

FIG. 8 shows the construction of pBinRi.

FIG. 9 shows the construction of the plant transformation vector pMDB2.

FIG. 10 shows the construction of pVT0.

FIG. 11 shows the construction of pVT1.

FIG. 12 shows the construction of the plant transformation vector pVT2.

DETAILED DESCRIPTION OF THE INVENTION

The following Examples illustrate the invention.

EXAMPLE 1

Extraction of basic heat-stable proteins from Mirabilis jalapa seeds

One kg of M jalapa seeds (purchased from Chiltern Seeds, Ulverston,Cumbria, UK) was ground in a coffee mill and the resulting meal wasextracted for 2 hours at 4° C. with 2 liters of an ice-cold extractionbuffer containing 10 mM NaH₂ PO₄, 15 mM Na₂ HPO₄, 100 mM KCl, 2 mM EDTA,2 mM thiourea, 1 mM PMSF and 1 mg/l leupeptin. The homogenate wassqueezed through cheesecloth and clarified by centrifugation (5 min at5,000×g). Solid ammonium sulphate was added to the supernatant to obtain35% relative saturation and the precipitate formed after standing for 1hour at room temperature was removed by centrifugation (10 min at5,000×g). The supernatant was adjusted to 65% relative ammonium sulphatesaturation and the precipitate formed overnight at room temperaturecollected by centrifugation (30 min at 5,000×g). After redissolving thepellet in 300 ml 10 mm sodium phosphate buffer (pH 6) the solution washeated at 75° C. for 10 min. The coagulated insoluble material wasdiscarded after centrifugation (20 min at 5,000×g) and the supernatantwas dialyzed extensively against distilled water using benzoylatedcellulose tubing (Sigma) with a molecular weight cut off of 2,000 Da.After dialysis the solution was adjusted to 50 mM Tris-HCl (pH 9) byaddition of the ten-fold concentrated buffer, and subsequently passedover a Q-Sepharose Fast Flow (Pharmacia) column (12×5 cm) in equilibriumwith 50 mM Tris-HCl (pH 9). The proteins in the unbound fraction wereprecipitated by addition of ammonium sulphate to 75% relativesaturation. The precipitate was collected by centrifugation (20 min at5,000×g) and the pellet redissolved in 15 ml phosphate buffered saline(PBS). This material represents the basic heat-stable protein fractionof M jalapa seeds, and was the starting material used for the isolationand purification of the M jalapa antimicrobial proteins.

EXAMPLE 2

Antifungal activity assay

Antifungal activity was measured by microspectrophotometry as previouslydescribed (Broekaert, WF et al; 1990; FEMS Microbiol Lett, 69, 55-60).Routinely, tests were performed with 20 μl of a (filter-sterilized) testsolution and 80 μl of a fungal spore suspension (2×10⁴ spores/ml) inhalf strength Potato Dextrose Broth (Difco). Control microculturescontained 20 μl of (sterile) distilled water and 80 μl of the fungalspore suspension. Unless otherwise stated the test organism was Fusariumculmorum and incubation was done at 25° C. for 48 hours. Percent growthinhibition is defined as 100 times the ratio of the corrected absorbanceof the control microculture minus the corrected absorbance of the testmicroculture over the corrected absorbance at 595 nm of the controlmicroculture. The corrected absorbance values equal the absorbance at595 nm of the culture measured after 48 hours minus the absorbance at595 nm measured after 30 min. Values of growth inhibition lower than 10%are not indicated on the chromatograms.

EXAMPLE 3

Purification of antimicrobial proteins from M jalapa seeds

The starting material for the isolation of the M jalapa antimicrobialproteins was the basic heat-stable protein fraction extracted from themature seeds. FIG. 1A illustrates how the antimicrobial proteins werepurified by gel filtration chromatography. One ml fractions were appliedon a Superose-12 column (50×1.6 cm) previously equilibrated with PBS.Running buffer was PBS and the flow rate 1 ml/min. The eluate wasmonitored for absorbance at 280 nm and collected in 2.5 ml fractions ofwhich 2 μl was used in the microspectrophotometric antifungal activityassay described in Example 2 (results shown in the upper panel of FIG.1A). Upon fractionation, the mixture resolved into three peaks, wherebythe antifungal activity coeluted with the most retarded peak. Thefractions containing antifungal activity (material from this peak) werepooled, dialyzed against 50 mM Na-MES (pH5) and subjected to cationexchange chromatography. Active fractions combined from three parallelgel filtration chromatographic runs were loaded on a S-Sepharose HighPerformance column (10×1.6 cm) in equilibrium with 50 mm Na-MES (pH 5).The column was eluted at 3 ml/min with a linear gradient from 0 to 150mM NaCl in 50 mM Na-MES (pH 5), 450 ml total. The eluate was monitoredfor protein by measurement of the absorbance at 280 nm (results shown inthe lower panel of FIG. 1B) and collected in 15 ml fractions of which 2μl was tested in the microspectrophotometric antifungal activity assay(results shown in the upper panel of FIG. 1B). No antifungal activitywas found in the unbound fraction. Application of a linear NaCl gradientallowed the separation of two factors with antifungal activity. Thefirst factor, called Mj-AMP1 (Mirabilis jalapa--Antimicrobial Protein 1)eluted as a major peak around 50 mM NaCl, whereas the second factor,designated analogously as Mj-AMP2, eluted at 100 mM NaCl.

The purity of the isolated antimicrobial factors was verified byreverse-phase chromatography. HPLC profiles of the purified Mj-AMPs wereobtained by loading two hundred μg amounts of Mj-AMP1 and of Mj-AMP2 ona Pep-S (porous silica C₂ /C₁₈) column (25×0.4 cm) (Pharmacia) inequilibrium with 0.1% TFA. The column was eluted at 1 ml/min with thefollowing gradients (solvent B is methanol containing 0.1 % TFA): 0-1min, 0% B; 1-3 min, 0-30% B; 3-23 min, 30-80% B; 23-25 min, 80-100% B.The eluate was monitored for protein by measurement of the absorption at280 nm. One ml fractions of the eluate were collected, freeze-dried, andfinally dissolved in 100 μl distilled water of which 20 μl was used in amicrospectrophotometric antifungal activity assay. Chromatography wasperformed on a Waters 600 HPLC station.

FIG. 2A and FIG. 2B show the HPLC profiles of purified Mj-AMP1 andMj-AMP2 respectively. The lower panels show monitoring of the eluate forprotein by measurement of the absorption at 280 nm. Results of themicrospectrophotometric antifungal activity assay are shown in the upperpanels. Both Mj-AMP1 and Mj-AMP2 yielded single, well resolved, majorpeaks that coeluted exactly with the antifungal activity.

EXAMPLE 4

Molecular structure of the purified antimicrobial proteins

The molecular structure of the Mj-AMPs was further analysed.Electrophoresis was performed on precast commercial gels (PhastGel HighDensity from Pharmacia) using a PhastSystem (Pharmacia) electrophoresisapparatus. The sample buffer contained 200 mM Tris-HCl (pH 8.3), 1%(w/v) SDS, 1 mM EDTA, 0.005% bromophenol blue and, unless otherwisestated, 1% (w/v) dithiothreitol (DTT). Silver staining of proteins inthe gel was done according to Development Technique File no 210 ofPhastSystem (Pharmacia LKB Biotechnology, Uppsala, Sweden), anddiffusion blotting followed by silver staining of the blots wasperformed as described in Development Technique File no 222 ofPhastSystem.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) ofthe unreduced mj-AMPs failed to reveal the presence of proteins.However, when nitro-cellulose blots were prepared from the gels, Mj-AMP1and Mj-AMP2 appeared on the blots as single bands with molecular weightsof 8 kDa and 7 kDa, respectively. Unreduced protein samples (200 ng)were dissolved in sample buffer without DTT, separated on PhastGel HighDensity (Pharmacia), blotted on to nitrocellulose and silver-stained onthe blot. Myoglobin fragments were used as molecular weight markers. Thereduced Mj-AMPs could be stained directly in the gel upon SDS-PAGE,showing rather diffuse bands in the apparent molecular weight zone of 3to 4 kDa. Protein samples (200 ng) were reduced in sample buffercontaining DTT, separated on PhastGel High Density and silver-stained inthe gel.

It appears therefore that the antimicrobial factors are dimeric proteinsstabilised by disulphide bridges, each comprised of two identicalsubunits (about 4 kDa and 3.5 kDa for Mj-AMP1 and Mj-AMP2,respectively). Attempts to determine the molecular weight of the nativeMj-AMPs by gel filtration on either Superose-12 or Superdex yieldedclearly underestimated values (between 1 and 2 kDa), most probably dueto interactions with the gel matrices.

Periodic Acid Schiff's (PAS) staining of reduced Mj-AMPS separated bySDS-PAGE was negative, suggesting that their polypeptides arenon-glycosylated. PAS staining for glycoproteins was done by the methodof Zacharius, RM et al (1969; Anal Biochem, 30, 148-152) using ovalbumin(3.2% carbohydrate) as a positive control sample. The pI values ofMj-AMP1 and Mj-AMP2 were determined by isoelectric focusing and found tobe about 10.5 for both proteins. Isoelectric focussing was done onprecast Immobiline Dry Strips (Pharmacia) using marker proteins in thepI range from 4.7 to 10.6 (Pharmacia).

All cysteine residues of mj-AMP2 appeared to participate in disulphidebonds, as unreduced Mj-AMP2 did not contain free thiol groups. Likewise,Mj-AMP1 only reacted with thiol reagents in its reduced but not in itsunreduced state. Thiol group determination was done by thedithionitrobenzoic acid method of Ellman, GL (1959; Arch BiochemBiophys, 82, 70-74) using 10 nmol of protein. Reduced protein sampleswere prepared by reaction with 10 mM DTT for 1 hour at 45° C. followedby extensive dialysis against distilled water using benzoylatedcellulose tubing (Sigma) with a molecular weight cut off of 2 kDa.

EXAMPLE 5

Amino acid sequencing of the MJ-AMPs

Cysteine residues of antimicrobial proteins were modified byS-carboxyamidomethylation as follows: 100 μg amounts of purifiedproteins were dissolved in 150 μl 0.3 M Tris-HCl (pH 8.6) containing 30mM DTT and reacted for 1 hour at 45° C. iodoacetamide was added to afinal concentration of 100 mN and the mixture was kept in the dark at37° C. for 1 hour. The reaction was finally quenched by addition of DTTto a final concentration of 100 mM and allowed to react for anadditional hour at 37° C. Desalting was done by High Performance LiquidChromatography (HPLC) on a Pep-S (porous silica C₂ /C₁₈) (Pharmacia)column (25×0.4 cm). The carboxyamidomethylated proteins were recoveredby eluting the column with a linear gradient from 0.1% trifluoroaceticacid (TFA) to 2-propanol containing 0.1% TFA. The resulting proteinfractions were subjected to amino acid sequence analysis in a 477AProtein Sequencer (Applied Biosystems) with on-line detection ofphenylthiohydantoin amino acid derivatives in a 120A Analyser (AppliedBiosystems).

Sequence data were obtained for 37 residues of Mj-AMP1, after treatmentwith pyroglutamate amino peptidase (to remove a blocked N-terminalresidue) and with trypsin (to generate internal peptides). The molecularmass of Mj-AMP1 was verified by fast atom bombardment mass spectroscopyand found to be 3976 daltons, corresponding exactly to the predictedmolecular mass. The sequence was homologous with Mj-AMP2, featuringdifferences at amino acid 28 (an asparagine replaced by a glycine),amino acid 33 (a valine replaced by a tyrosine), and amino acid 35 (anarginine replaced by a lysine). A glutamine at amino acid 1 was furtheridentified in Mj-AMP1. Both peptides contain 6 cysteine residues. FIG.3A shows the complete amino acid sequences of Mj-AMP1 and Mj-AMP2;differences are indicated by arrows.

The Mj-AMP sequences were found to be homologous to neurotoxins found inthe venom of invertebrates including μ-agatoxins from the spiderAgelenopsis aperta (Skinner, WS et al; 1989; J Biol Chem, 264,2150-2155), conotoxins from marine snail Conus sp. (Olivera, BM, et al;1985, Science, 230, 1338-1343), toxins from the Buthotus scorpion (FazalA et al, 1989, FEBS Letters 257 260-2), and curtatoxins (Stapleton etal, 1990, J Biol Chem, 265(4), 2054-2059). FIG. 3B shows the amino acidsequences of Mj-AMP1 and Mj-AMP2, aligned with those of the neurotoxins.Homologous amino acids are boxed. Dashes indicate gaps introduced foroptimal alignment of the sequences.

EXAMPLE 6

Stability of the antifungal activity of the Mj-AMPs

Table 1 summarises the results of further testing of the stability ofthe antifungal activity of the Mj-AMPs. Tests for antifungal activitywere performed with 20 μl samples diluted five-fold with growth mediumcontaining Fusarium culmorum spores (2×10⁴ spores/ml), according to theassay method given in Example 2. Control samples contained eitherMj-AMP1 at 500 μg/ml or Mj-AMP2 at 100 μg/ml in 10 mM sodium phosphatebuffer (pH 7). Reduction was done by addition of DTT at 2.5 mM followedby incubation at 37° C. for 2 hours. pH stability was tested in thefollowing buffers: 20 mM glycine-HCl (pH 2 and 3); 20 mMdiethanolamine-HCl (pH 10); and 20 mM glycine-NaOH (pH 11). After 1 hourof incubation in the appropriate buffers the samples were dialyzed for16 hours against 10 mm sodium phosphate buffer (pH 7). For digestions,proteases were added at 100 μg/ml and incubated at 37° C. for 16 hours.

                  TABLE 1                                                         ______________________________________                                        Stability of the antifungal activity of Mj-AMPs                                                 Relative antifungal                                                           activity (shown as % of                                                       control activity)                                           Treatment         Mj-AMF1        Mj-AMP2                                      ______________________________________                                        Control           100            100                                          Reduction         <6             <3                                           80° C., 10 min                                                                           100            100                                          90° C., 10 min                                                                           100            100                                          100° C., 10 min                                                                          100            100                                          pH 2, 60 min      100            100                                          pH 3, 60 min      100            100                                          pH 10, 60 min     100            100                                          pH 11, 60 min     100            100                                          Pronase E digestion                                                                             50             25                                           Chymotrypsin digestion                                                                          12             50                                           Trypsin digestion 75             100                                          Proteinase K digestion                                                                          100            100                                          ______________________________________                                    

After reduction of their disulphide bonds, Mj-AMPs completely lost theirantifungal activity against F culmorum. The activity of the nativeproteins, however, was not affected by heat treatments at up to 100° C.for 10 minutes, and was stable over the pH range of 2 to 11. Mj-AMP1 wassensitive to the protease chymotrypsin, but resisted almost completelydigestion by pronase E, trypsin and proteinase K, whereas Mj-AMP2 wasmost sensitive to pronase E treatment.

EXAMPLE 7

Antifungal potency of the Mj-AMPs

The antifungal potency of the Mj-AMPs was examined on 13 different plantpathogenic fungi, and compared to that of two known antifungal proteins,Urtica dioica agglutinin or UDA (Broekaert, WF et al; 1989; Science,245, 1100-1102) and β-purothionin (Hernandez-Lucas, C et al; 1974; ApplMicrobiol, 28, 165-168). Fungi were grown on six cereal agar under whitefluorescent light and spores were harvested and stored as previouslydescribed (Broekaert, WF et al; 1990; FEMS Microbiol Lett, 69, 55-60).The following fungal strains were used: Alternaria brassicola MUCL20297, Ascochyta pisi MUCL 30164, Botrytis cinerea MUCL 30158,Colletotrichum lindemuthianum MUCL 9577, Fusarium culmorum IMI 180420,Fusarium oxysporum f.sp. pisi IMI 236441, Fusarium oxysporum f.sp.lycopersici MUCL 909, Nectria haematococca Collection Van Etten 160-2-2,Phoma betae MUCL 9916, Pyrenophora tritici-repentis MUCL 30217,Pyricularia oryzae MUCL 30166,Venturia inaegualis MUCL 15927,Verticillium dahliae MUCL 19210. UDA was isolated from stinging nettle(Urtica dioica) rhizomes as previously described (Peumans, WJ et al;1983; FEBS Lett, 177, 99-103). The β-purothionin was purified from wheatendosperm by the method of Redman, DG and Fisher, N (1969; J Sci FdAgric, 20, 427-432).

Table 2 summarises the results. Serial dilutions of Mj-AMP1, Mj-AMP2,UDA and β-purothionin were applied to fungi and the percent growthinhibition measured by microspectrophotometry (as described in Example2). The concentration required for 50% growth inhibition after 48 hoursof incubation was taken as the IC₅₀ value, which was calculated from thedose-response curves. The IC₅₀ of the slow growing fungus Venturiainaequalis was measured after 10 days of incubation.

The concentrations required for 50% inhibition of fungal growth after 48hours of incubation (IC₅₀) varied from 6 to 300 μg/ml for Mj-AMP1, from0.5 to 20 μg/ml for Mj-AMP2, from 0.5 to 15 μg/ml for β-purothionin, andfrom 20 to over 1,000 μg/ml for UDA depending on the test organism. Onan average basis the obtained antifungal activity series is as follows:Mj-AMP2=β-purothionin>Mj-AMP1>UDA. Some fungi, such as B cinerea, Clindemuthianum and V inaequalis, are clearly more sensitive to Mj-AMP2than to β-purothionin. Conversely, the latter protein is most effectivein deterring growth of other fungi such as F oxysporum f.sp. pisi and Ptritici-repentis.

With all tested antifungal proteins, the extent of growth inhibitiontended to decrease as the incubation time increased. For instance, theIC₅₀ value of Mj-AMP1 on C lindemuthianum rose from 6 μg/ml after 48hours of incubation to 12 μg/ml after 72 hours. The time-dependent dropin antifungal activity, however, was less pronounced for Mj-AMP2 andβ-purothionin than for Mj-AMP1 or UDA. Also, Mj-AMP2 and β-purothionincharacteristically produced steeper dose-response curves than Mj-AMP1 orUDA. FIG. 4 shows the time-dependent growth inhibition curves of thefungi Colletotrichum lindemuthianum (panels A, C, E, G) and Alternariabrassicola (panels B, D, F, H) measured at varying concentrations of thefollowing proteins: Mj-AMP1 (panels A and B), Mj-AMP2 (panels C and D),UDA (panels E and F), and β-purothionin (panels G and H). The percentgrowth inhibition was recorded after 48 h ( . . . ), after 60 h (0 . .. 0) or after 72 h (▪ . . . ▪).

                  TABLE 2                                                         ______________________________________                                        Antifungal activity of MJ-AMPS and other antifungal proteins                  on different phytopathogenic fungi                                                      IC.sub.50 (μg/ml)                                                                                       β-puro-                           Fungus      Mj-ANP1   Mj-AMP2  UDA     thionin                                ______________________________________                                        A   brassicola  20        6      200     3                                    A   pisi        200       6      1,000   3                                    B   cinerea     60        2      >1,000  12                                   C   lindemuthianum                                                                            6         1       20     15                                   F   culmorum    30        3      >1,000  1                                    F   oxysporum   15        5      >1,000    0.5                                    f.sp. pisi                                                                F   oxysporum   200       10     *ND     ND                                       f.sp. lycopersici                                                         N   haematococca                                                                              15        0.5    200     1                                    P   betae       25        6       50     4                                    P   tritici-repentis                                                                          300       20     200     4                                    P   oryzae      6         0.5    ND      ND                                   V   dahliae     12        0.5     80       0.5                                V   inaequalis  12        1      1,000   5                                    ______________________________________                                         * ND = not determined                                                    

EXAMPLE 8

Antifungal activity of the Mj-AMPs against foliar diseases: in vivo test

The basic heat-stable protein fraction from Mirabilis jalapa and a puresample of Mj-AMP1 were tested against foliar fungal diseases of plantsusing the following technique.

The plants were grown in John Innes Potting Compost (No 1 or 2) in 4 cmdiameter mini-pots. The protein preparation was formulated immediatelyprior to use by dissolving in sterile deionised water and diluting tothe appropriate concentration. It is assumed that the protein extractcontains approximately 1% active ingredient. A pure sample of Mj-AMP1was similarly prepared. The formulations were applied to the plants as afoliar spray. The spray was applied to maximum discrete dropletretention. Tween 20, to give a final concentration of 0.05%, was addedwhen the spray was applied to cereals. The protein preparation wasapplied to the foliage (by spraying) one or two days before the plantwas inoculated with the disease (protectant application). Foliarpathogens were applied by spray as spore suspensions on to the leaves oftest plants. After inoculation, the plants were put into an appropriateenvironment to allow infection to proceed and then incubated until thedisease was ready for assessment. The period between inoculation andassessment varied from four to fourteen days according to the diseaseand environment.

Results are shown in Table 3 for the three fungal pathogens: Septorianodorum (Fungi Imperfecti) tested on wheat, Plasmopara viticola(Phycomycete) tested on grapevine, and Cercospora beticola tested onsugarbeet. The disease control was recorded by the following grading:4=no disease; 3=trace-5% of disease on untreated plants; 2=6-25% ofdisease on untreated plants; 1=26-60% of disease on untreated plants;0=61-100% of disease on untreated plants.

                  TABLE 3                                                         ______________________________________                                        Control of fungal diseases in vivo by Mirabilis                               proteins                                                                                 FOLIAR SPRAY CONC.   Mj-AMP1                                                  CRUDE PROTEIN EXTRACT                                                                              100 μg/ml                                  TEST ORGANISM                                                                            10 mg/ml  2.5 mg/ml                                                                              Untreated                                                                             control                                 ______________________________________                                        S nodorum  2-3       0        0       --                                      P viticola 4         4        0       --                                      C beticola --        --       0       3                                       ______________________________________                                    

These results confirm that the Mirabilis protein extract or the purifiedMj-AMP1 peptide can act as a fungicide in vivo when applied as a foliarspray.

EXAMPLE 9

Antifungal activity of the Mj-AMPs against foliar diseases: in vitrotest

The protein preparation as defined in Example 8 was also tested foractivity against a spectrum of fungal pathogens in vitro. A measuredaliquot of the formulated material was dispersed in an agar medium(Petris minimal medium, consisting of a salts solution of 2 g Ca(NO₃)₂,0.725 g MgSO₄, 0.725 g KH₂ PO₄, 0.6 g KCl, 17.2 g NaH₂ PO₄, 17.725 g Na₂HPO₄ in 1000 ml of water, plus an agar solution consisting of 10 gTechnical Agar no 3 (Oxoid) and 50 g sucrose in 800 ml water). The agarwas subsequently inoculated with a range of pathogens using either sporesuspensions or mycelial plugs. The agar plates were then inoculated fora period of up to 5 days before assessment.

Results are shown in Table 4. The disease control was recorded by thefollowing grading: 4=no growth of pathogen (complete inhibition);3=trace growth of pathogen; 2=restricted/moderate growth of pathogen;0=no inhibition of pathogen; M=missing result.

                  TABLE 4                                                         ______________________________________                                        Control of fungal diseases in vitro by Mirabilis                              protein extract                                                                              CONCENTRATION                                                                 OF CRUDE                                                                      PROTEIN EXTRACT Untreated                                      TEST ORGANISM  10 mg/ml  2.5 mg/ml control                                    ______________________________________                                        Penicillium pinophilum                                                                       4         2         0                                          Aureobasidium pullulans                                                                      4         2         0                                          Aspergillus niger                                                                            2         3         0                                          Penicillium digitatum                                                                        4         4         0                                          Colletotrichum musae                                                                         2         2         0                                          Botrytis cinerea                                                                             4         3         0                                          Fusarium culmorum                                                                            4         4         0                                          Geotrichium candidum                                                                         4         3         0                                          Verticillium albo-atrum                                                                      M         2         0                                          ______________________________________                                    

The protein extract shows a broad spectrum of antifungal activity atboth rates tested.

EXAMPLE 10

Inhibitory activity of Mj-AMPs against yeast

The inhibitory activity of Mj-AMPs against yeast, Saccharomycescerevisiae was tested. The results shown in Table 5 indicate thatMj-AMPs do inhibit the growth of yeast at concentrations of 500 μg/ml orabove.

                  TABLE 5                                                         ______________________________________                                        Inhibitory activity of purified Mj-AMPS                                               CONCENTRATION (μg/ml)                                                      without Ca/K  with Ca/K                                                       500   50      5       500   50    5                                   ______________________________________                                        Mj-AMP1   +++     +++     +     +++   -     -                                 Mj-AMP2   +++     +++     +++   +++   +++   -                                 ______________________________________                                         +++:complete inhibition                                                       +:some inhibition                                                             -:no inhibition                                                          

EXAMPLE 11

Antibacterial activity of the Mj-AMPs: in vitro test

The antibacterial activity of the Mj-AMPs was tested against a range ofgram-positive and gram-negative bacteria: Bacillus megaterium, Sarcinalutea, Escherichia coli and Erwinia carotovora. β-purothionin and UDAwere also tested for comparison (see Example 7). Tests were performed insoft agarose medium containing 1% tryptone and, with or without 1 mMCaCl₂ and 50 mM KCl. Absorbance, 595 nm, of the culture was measuredafter 48 hours. Results are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        Antibacterial activity of Mj-AMPS, β-purothionin and UDA                          IC.sub.50 (μg/ml)                                                 Bacterium  Mj-AMP1   Mj-AMP2    β-pt                                                                            UDA                                    ______________________________________                                        MEDIUM -Ca/K                                                                  B megaterium                                                                              6         2          1      250                                   S lutea    100       50          8     >500                                   E coli     >500      >500       200    >500                                   E carotovora                                                                             >500      >500       >500   >500                                   MEDIUM +Ca/K                                                                  B megaterium                                                                              20       10          1     >500                                   S lutea    >500      >500        20    >500                                   E coli     >500      >500       >500   >500                                   E carotovora                                                                             >500      >500       >500   >500                                   ______________________________________                                    

The results show that while the Mj-AMPs appear to have no activityagainst gram-negative bacteria, they do inhibit growth of gram-positivebacteria.

EXAMPLE 12

Molecular cloning and sequence of the Mj-AMP cDNAs

Fully matured seeds of mirabilis jalapa were collected from outdoorgrown plants, immediately frozen in liquid nitrogen and stored at -80°C. Total RNA was extracted from 15 g of pulverised seeds by the methodof De Vries et al (1988, Plant Molecular Biology Manual, B6, 1-13). Poly(A)⁺ RNA was purified by oligo (dT)-cellulose affinity chromatography asdescribed by Silflow et al (1979, Biochemistry, 18, 2725-2731) yieldingabout 10 g of poly (A)⁺ RNA. Double-stranded cDNAs were prepared from 2g of poly (A)⁺ RNA according to Gubler and Hoffman (1983, Gene, 25,263-269) using the cDNA Synthesis System Plus of Amersham. The cDNAswere cloned into the λgt10 phage vector (Amersham) after ligation toEcoRI linkers (Amersham) according to the manufacturer's instructions.Phage DNA was packaged in vitro with the Gigapack II Gold packagingsystem (Stratagene).

A DNA probe for screening of the cDNA library was produced by polymerasechain reaction (PCR) as follows. Two degenerate oligonucleotides weresynthesised: OWB3 (5'TGYATHGGNAAYGGNGGNMGNTG) and OWB4(5'ACNCCRTANCCYTGRTTNGGYTG). OWB3 corresponds to amino acids 1 to 8 ofMj-AMP2 and has a sense orientation. OWB4 corresponds to amino acids 25to 32 of Mj-AMP2 and has an antisense orientation. PCR was performedwith the Taq polymerase under standard conditions (Sambrook et al, 1989,Molecular Cloning, Cold Spring Harbour Lab Press) using OWB3 and OWB4 asamplimers and 25 ng of cDNA as target DNA. The temperature programmeincluded an initial step at 94° C. for 5 min. 30 cycles (94° C. for 1min; 45° C. for 2 min; 72° C. for 3 min) and a final step at 72° C. for10 min. The 100 bp PCR amplification product was purified on a 3%agarose (NuSieve, FMC) gel and reamplified by PCR under the sameconditions except that the reaction mixtures contained 130 μM dTTP and70 μM digoxigenin-11-dUTP instead of 200 μM dTTP. Thedigoxigenin-labelled PCR product was purified on a 3% NuSieve agarosegel.

About 100,000 plaque forming units of the λgt10 cDNA library werescreened with the digoxigenin-labelled PCR product by in situ plaquehybridisation using nylon membranes (Hybond -N, Amersham). Membraneswere air-dried and DNA was crosslinked on the membranes under UV light(0.15 J/cm²). Hybridisation was performed for 16 hours at 68° C. in5×SSC, 1% blocking reagent (Boehringer Mannheim), 0.1%N-lauroylsarcosine, 0.02% sodium dodecylsulphate containing 10 ng/ml ofheat denatured digoxigenin-labelled probe. Non-specifically bound probewas removed by rinsing twice for 5 min in 2×SSC/0.1% SDS at 25° C. andtwice for 15 min in 0.1×SSC/0.1% SDS at 68° C. Detection of the probewas done using anti-digoxigenin antibodies linked to alkalinephosphatase (Boehringer Mannheim) and its substrate5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim) according tothe manufacturer's instructions. Positive plaques were purified by twoadditional screening rounds with the same probe under the sameconditions. Inserts from purified plaques were subcloned into the EcoRIsite of pEMBL18 (Dente et al, 1983, Nucl Acid Res, 11, 1145-1155).Nucleotide sequencing was done with an ALF automated sequencer(Pharmacia) using fluoresceine-labelled M13 forward and reverse primers(Pharmacia). Sequence analysis was performed by the PC-gene software(Intelligenetics).

Inserts from two positive clones, MJ1 and MJ2 were subjected tonucleotide sequence analysis.

MJ1 is 360 nucleotides in length and appears to be truncated at its 5'end. The coding region contains 61 amino acids including the 37 aminoacids of Mj-AMP1 at the carboxy-terminal part. Again, the amino-terminalpart (24 amino acids) has all the features of a signal peptide but istruncated since it lacks an initial methionine. The 3' untranslatedregion is 172 nucleotides long and includes a putative polyadenylationsignal (AATAAG) at position 326 and a 12-nucleotide poly (A) tail.

MJ2 is 433 nucleotides long and contains an open reading frame of 63amino acids. The 36 carboxy-terminal amino acids correspond exactly tothe amino acid sequence of Mj-AMP2, whereas the 27 amino-terminal aminoacids have a predicted signal peptide structure obeying the (-1,-3)-rule (von Heijne, 1985, Mol Biol, 184, 99-105). MJ2 has34-nucleotide and 210-nucleotide untranslated regions at the 5' and 3'end, respectively. A putative polyadenylation signal (AATAAG) is locatedat position 399 and is followed 11 nucleotides downstream by an18-nucleotide poly (A) tail.

FIGS. 5 and 6 show the nucleotide sequences and deduced amino acidsequences of clones MJ1 and MJ2, respectively. The open boxes correspondto the amino acid sequence of the mature Mj-AMPs. Stop codons are markedwith asterisks and the potential polyadenylation sites are underlined.

EXAMPLE 13

Construction of the expression vector pMDB1

The insert MJ2 (containing the Mj-AMP2 sequence) was removed by BamHIdigestion from the pEMBL18⁺ vector and sub-cloned in a BamHI site of theexpression vector pFAJ3002. pFAJ3002 is a modification of the expressionvector pFF19 (Zimmermans et al, 1990, J Biotechnology, 14, 333-344)comprising a HindIII substitution of an EcoRI site and a CaMV35S doubleenhancer sequence. A clone comprising MJ2 in a sense orientation wasdesignated pMDB1: its construction is shown in FIG. 7.

EXAMPLE 14

Construction of the plant transformation vector pMDB2 for extracellularexpression of Mj-AMP2

The MJ2 CaMV35S promoter insert was HindIII digested from pMDB1 andsubcloned into the unique HindIII site of pBinRi. pBinRi is a modifiedversion of the plant transformation vector pBin19 (Bevan, 1984, NucleicAcids Research, 12:22, 8711-8721) wherein the unique EcoR1 and HindIIIsites are switched and a wild type npt II gene is included asillustrated in FIG. 8. The new plant transformation vector is designatedpMDB2 and is illustrated in FIG. 9.

EXAMPLE 15

Construction of the plant transformation vector pVT2 for vacuolarexpression of Mj-AMP2

A construct was made to ensure the proper processing and transport ofMj-AMP2 to the vacuoles of transgenic plants. A nucleotide sequencecoding for a 15 amino acid propeptide (Bednarek S.Y. et al, 1991, PlantCell 3, 1195-1206) with an additional amino acid to facilitate cleavageof the propeptide from the mature protein was synthesised. Using PCRtechniques, the new synthetic sequence for the propeptide was linked tothe plant cDNA encoding the Mj-AMP2 peptide and signal peptide (MJ2) togive the insert designated VT, containing a KpnI site at the 5' end anda PstI site at the 3' end. The KpnI-PstI fragment (generated by PCR) wascloned in the KpnI-PstI site of pEMBL18⁺ to give pVT0, shown in FIG. 10.The KpnI-PstI fragment of pVT0 was cloned in the KpnI-PstI sites of theplant expression cassette pFF19 to give pVT1, shown in FIG. 11. TheHindIII-EcoRI fragment of pVT1 was then cloned in the HindIII-EcoRI siteof pBinRi to give the plant transformation vector designated pVT2, shownin FIG. 12.

EXAMPLE 16

Plant Transformation

Agrobacterium strain LBA4404 ACH5 pAL4404! was transformed to containeither of the vectors pMDB2 or pVT2, using the method of de Framond A etal (Biotechnology, 1, 262-9).

Tobacco transformation was carried out using leaf discs of Nicotianatabacum Samsun based on the method of Hozsch RB et al (1985, Science,227, 1229-31) and co-culturing with Agrobacterium strains containingpMDB2 or pVT2. Co-cultivation was carried out under selection pressureof 100 μg/ml kanamycin.

Transgenic plants (transformed with pMDB2 or pVT2) were regenerated.These transgenic plants are being analysed for expression of the newlyintroduced genes using standard Western blotting techniques. Plantscapable of constitutive expression of the-introduced genes will beselected and self-pollinated to give seed. F1 seedlings of thetransgenic plants will be further analysed.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 13                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 37 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: MJ-AMP1 F - #IGURE 4A                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Gln Cys Ile Gly Asn Gly Gly Arg Cys Asn Gl - #u Asn Val Gly Pro Pro         #                15                                                           - Tyr Cys Cys Ser Gly Phe Cys Leu Arg Gln Pr - #o Gly Gln Gly Tyr Gly         #            30                                                               - Tyr Cys Lys Asn Arg                                                                 35                                                                    - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 36 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: MJ-AMP2 F - #IGURE 4A                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Cys Ile Gly Asn Gly Gly Arg Cys Asn Glu As - #n Val Gly Pro Pro Tyr         #                15                                                           - Cys Cys Ser Gly Phe Cys Leu Arg Gln Pro As - #n Gln Gly Tyr Gly Val         #            30                                                               - Cys Arg Asn Arg                                                                     35                                                                    - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 37 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: MJ-AMP1 F - #IGURE 4B                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Gln Cys Ile Gly Asn Gly Gly Arg Cys Asn Gl - #u Asn Val Gly Pro Pro         #                15                                                           - Tyr Cys Cys Ser Gly Phe Cys Leu Arg Gln Pr - #o Gly Gln Gly Tyr Gly         #            30                                                               - Tyr Cys Lys Asn Arg                                                                 35                                                                    - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 36 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: MJ-AMP2 F - #IGURE 4B                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Cys Ile Gly Asn Gly Gly Arg Cys Asn Glu As - #n Val Gly Pro Pro Tyr         #                15                                                           - Cys Cys Ser Gly Phe Cys Leu Arg Gln Pro As - #n Gln Gly Tyr Gly Val         #            30                                                               - Cys Arg Asn Arg                                                                     35                                                                    - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 34 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (vi) ORIGINAL SOURCE:                                                   #GS FIGURE 4B ORGANISM: CONOTOXIN                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Ala Cys Ser Gly Arg Gly Ser Arg Cys Pro Pr - #o Gln Cys Cys Met Gly         #                15                                                           - Leu Arg Cys Gly Arg Gly Asn Pro Gln Lys Cy - #s Ile Gly Ala His Glu         #            30                                                               - Asp Val                                                                     - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 25 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (vi) ORIGINAL SOURCE:                                                   #MV2A FIGURE 4BRGANISM: CONOTOXIN                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Cys Lys Gly Lys Gly Ala Ser Cys Arg His Th - #r Ser Tyr Asp Cys Cys         #                15                                                           - Thr Gly Ser Cys Asn Arg Gly Lys Cys                                         #            25                                                               - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 38 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (vi) ORIGINAL SOURCE:                                                   #3 FIGURE 4B) ORGANISM: AGATOXIN                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Ala Asp Cys Val Gly Asp Gly Gln Arg Cys Al - #a Asp Trp Ala Gly Pro         #                15                                                           - Tyr Cys Cys Ser Gly Tyr Tyr Cys Ser Cys Ar - #g Ser Met Pro Tyr Cys         #            30                                                               - Arg Cys Arg Ser Asp Ser                                                             35                                                                    - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 38 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (vi) ORIGINAL SOURCE:                                                   #2 FIGURE 4B) ORGANISM: CURTATOXIN                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Ala Asp Cys Val Gly Asp Gly Gln Lys Cys Al - #a Asp Trp Phe Gly Pro         #                15                                                           - Tyr Cys Cys Ser Gly Tyr Tyr Cys Ser Cys Ar - #g Ser Met Pro Tyr Cys         #            30                                                               - Arg Cys Arg Ser Asp Ser                                                             35                                                                    - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 28 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: BUTHUS PE - #P 2 FIGURE 4B                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Val Gly Cys Glu Glu Asp Pro Met Asn Cys Ly - #s Gly Lys Gln Ala Lys         #                15                                                           - Pro Thr Cys Cys Asn Gly Val Cys Asn Cys As - #n Val                         #            25                                                               - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 61 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (vi) ORIGINAL SOURCE:                                                   #AMINO ACID SEQUENCESM: FIGURE 6                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Leu Pro Val Ala Phe Leu Lys Phe Ala Ile Va - #l Leu Ile Leu Phe Ile         #                15                                                           - Ala Met Ser Ala Met Ile Glu Ala Gln Cys Il - #e Gly Asn Gly Gly Arg         #            30                                                               - Cys Asn Glu Asn Val Gly Pro Pro Tyr Cys Cy - #s Ser Gly Phe Cys Leu         #        45                                                                   - Arg Gln Pro Gly Gln Gly Tyr Gly Tyr Cys Ly - #s Asn Arg                     #    60                                                                       - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 360 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                   #BASE SEQUENCEORGANISM: FIGURE 6                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - CTTCCCGTTG CCTTCCTCAA ATTCGCTATT GTGTTGATTC TCTTCATTGC CA - #TGTCCGCA         60                                                                          - ATGATAGAAG CACAATGCAT AGGAAATGGA GGAAGATGTA ACGAGAACGT GG - #GGCCACCA        120                                                                          - TACTGCTGCT CCGGTTTCTG CCTCCGTCAA CCTGGACAAG GTTATGGATA TT - #GTAAGAAC        180                                                                          - CGCTGAGCAA GAGCATGAAA GCAAGGCCAA TGTGTGGTCT ACTAATTTAG CC - #TCAAATGT        240                                                                          - TATTTATTTG CATGTCTTGT GTTTCTTAAT TACCTTCTTT GTGTCTAAGA AG - #GTATAGAT        300                                                                          - CAATAGTTTC TACTTTACTA CTATGAATAA GAGGCTTTGA TTTGGTTTAA AA - #AAAAAAAA        360                                                                          - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 63 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (vi) ORIGINAL SOURCE:                                                   #AMINO ACID SEQUENCE MJ-AMP2RE 7                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Met Ala Lys Val Pro Ile Ala Phe Leu Lys Ph - #e Val Ile Val Leu Ile         #                15                                                           - Leu Phe Ile Ala Met Ser Gly Met Ile Glu Al - #a Cys Ile Gly Asn Gly         #            30                                                               - Gly Arg Cys Asn Glu Asn Val Gly Pro Pro Ty - #r Cys Cys Ser Gly Phe         #        45                                                                   - Cys Leu Arg Gln Pro Asn Gln Gly Tyr Gly Va - #l Cys Arg Asn Arg             #    60                                                                       - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 433 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                   #BASE SEQUENCE MJ-AMP2: FIGURE 7                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - ATATCATTCA AATATACTAA ACTAATTATA AAAAATGGCT AAGGTTCCAA TT - #GCCTTTCT         60                                                                          - CAAATTCGTC ATCGTGTTGA TTCTCTTCAT TGCCATGTCA GGCATGATAG AA - #GCATGCAT        120                                                                          - AGGAAATGGA GGAAGATGTA ACGAGAACGT GGGCCCACCA TACTGCTGTT CG - #GGTTTCTG        180                                                                          - CCTCCGTCAA CCTAACCAAG GTTACGGTGT TTGCAGGAAC CGCTAATAAG CA - #AAGCCCAA        240                                                                          - AGTGTGGGTC ACAAAATAGT AGAGTTTAGC CTCAAATGTG GTTTATATAT GT - #AACAATCT        300                                                                          - TATATGTGTT TCTCTTGTGT TTCTTAATTA CCTTCTTTGT GTCTAAGAAG GT - #ATGGATAA        360                                                                          - ATAGTTTGTA CTTTACTATT ATGGTTTTTT CTTATATCAA TAAGAGGCTT TA - #ATTAAAAA        420                                                                          #     433                                                                     __________________________________________________________________________

We claim:
 1. A transformed biological system comprising a recombinant DNA system coding for an antimicrobial protein selected from the group consisting of the pure protein Mj-AMP1 having SEQ ID NO:1 and the pure protein Mj-AMP2 having SEQ ID NO:2, said system allowing expression of the encoded protein.
 2. A biological system as claimed in claim 1 which is a micro-organism.
 3. A biological system as claimed in claim 1 which is a plant. 